1. Field of the Invention
This invention relates to the field of medicine and tissue engineering, and in particular to drug releasing biodegradable implants.
2. Description of Related Art
Tissue engineering is a discipline wherein living cells are used to replace functional loss because of injury, disease, or birth defect in an animal or human. These replacement cells can be autologous, allogenic, or, in limited circumstances, xenogenic. The field of tissue engineering is a new area of medicine and optimal procedures have yet to be elucidated.
At present, there are several primary avenues investigators are using to engineer tissues. One is to harvest cells from a healthy donor, preferably from the same individual, or at least from an appropriate donor of the same species, and grow those cells on a scaffold in vitro. This scaffold is typically a three-dimensional polymer network, often composed of biodegradable fibers. Cells adherent to the polymer network can then typically be induced to multiply. This cell filled scaffold can be implanted into the impaired host with the goal that the cells will perform their physiological function and avoid destruction by the host immune system. To this end, it is important that purified cell lines are used, as the introduction of non-self immune cells can up-regulate a strong host immune attack. The difficulty with this approach is the scaffolding must be small, as no cell can survive more than a couple millimeters away from a source of oxygen and nutrients. Therefore, large scaffolds cannot be used, as the scaffold will not vascularize adequately in time to save the cells in the interior regions.
In another approach, an empty three-dimensional, biodegradable polymer scaffold is directly implanted in the patient, with the goal of inducing the correct type of cells from the host""s body to migrate into the polymer scaffold. The benefit is that vascularization can happen simultaneously with migration of cells into the matrix. A major problem is that there is currently no way to ensure that the appropriate cell types will migrate into the scaffold, and that the mechanical and biological properties will be maintained to provide the patient""s physiological need.
In both of the above approaches, the scaffold may be biodegradable, meaning that over time it will break down both chemically and mechanically. As this break down occurs, the cells secrete their own extracellular matrix, which plays a critical role in cell survival and function. In normal tissue, there is an active and dynamic reciprocal exchange between the constitutive cells of the tissue and the surrounding extracellular matrix. The extracellular matrix provides chemical signals that regulate the morphological properties and phenotypic traits of cells and may induce division, differentiation or even cell death. In addition, the cells are also constantly rearranging the extracellular matrix. Cells both degrade and rebuild the extracellular matrix and secrete chemicals into the matrix to be used later by themselves or other cells that may migrate into the area. It has also been discovered that the extracellular matrix is one of the most important components in embryological development. Pioneering cells secrete chemical signals that help following cells differentiate into the appropriate final phenotype. For example, such chemical signals cause the differentiation of neural crest cells into axons, smooth muscle cells or neurons.
The integrated relationship between extracellular matrix and tissue cells establishes the extracellular matrix as an important parameter in tissue engineering. If cells are desired to behave in a specific manner, then the extracellular matrix must provide the appropriate environment and appropriate chemical/biological signals to induce that behavior for that cell type. Currently it is not possible to faithfully reproducer a biologically active extracellular matrix. Consequently, some investigators use a biodegradable matrix that enables the cells to create their own extracellular matrix as the exogenous matrix degrades.
In the above-described approaches to tissue engineering, a polymer scaffolding provides not only the mechanical support, but also the three-dimensional shape that is desired for the new tissue or organ. Because cells must be close to a source of oxygen and nutrients in order to survive and function, a major current limitation is that of blood supply. Most current methodologies provide no specific means of actively assisting the incorporation of blood vessels into and throughout the polymer matrix. This places limitations on the physical size and shape of the polymer matrix. The only current tissue-engineering device that has made it into widespread clinical use is artificial skin, which by definition is of limited thickness. The present invention provides compositions and methods that promote the directed migration of appropriate cell types into the engineered extracellular matrix. By directing specific three-dimensional cell migration and functional patterns, directed vascularization can be induced, which overcomes the current limitations on the shape and size of polymer implants. It also ensures that appropriate cell types will be physically located in specific locations within the matrix. Compositions and methods are provided to modulate phenotypic expression as a function of both time and space.
The present invention provides tissue engineering compositions and methods wherein three-dimensional matrices for growing cells are prepared for in vitro and in vivo use. The matrices comprise biodegradable polymer fibers capable of the controlled delivery of therapeutic agents. The spatial and temporal distribution of released therapeutic agents is controlled by the use of predefined nonhomogeneous patterns of polymer fibers, which are capable of releasing one or more therapeutic agents as a function of time. The terms xe2x80x9cscaffold,xe2x80x9d xe2x80x9cscaffold matrixxe2x80x9d and xe2x80x9cfiber-scaffoldxe2x80x9d are also used herein to describe the three dimensional matrices of the invention. xe2x80x9cDefined nonhomogeneous patternxe2x80x9d in the context of the current application means the incorporation of specific fibers into a scaffold matrix such that a desired three-dimensional distribution of one or more therapeutic agents within the scaffold matrix is achieved. The distribution of therapeutic agents within the matrix fibers controls the subsequent spatial distribution within the interstitial medium of the matrix following release of the agents from the polymer fibers. In this way, the spatial contours of desired concentration gradients can be created within the three dimensional matrix structure and in the immediate surroundings of the matrix. Temporal distribution is controlled by the polymer composition of the fiber and by the use of coaxial layers within a fiber.
One aspect of the present invention is a biocompatible implant composition comprising a scaffold of biodegradable polymer fibers. In various embodiments of the present invention, the distance between the fibers may be about 50 microns, about 70 microns, about 90 microns, about 100 microns, about 120 microns, about 140 microns, about 160 microns, about 180 microns, about 200 microns, about 220 microns, about 240 microns, about 260 microns, about 280 microns, about 300 microns, about 320 microns, about 340 microns, about 360 microns, about 380 microns, about 400 microns, about 450 microns or about 500 microns. In various embodiments the distance between the fibers may be less than 50 microns or greater than 500 microns.
Additionally, it is envisioned that in various embodiments of the invention, the fibers will have a diameter of about 20 microns, about 40 microns, about 60 microns, about 80 microns, about 100 microns, about 120 microns, about 140 microns, about 160 microns, about 180 microns, about 200 microns, about 220 microns, about 240 microns, about 260 microns, about 280 microns, about 300 microns, about 320 microns, about 340 microns, about 360 microns, about 380 microns, about 400 microns, about 450 microns or about 500 microns (including intermediate lengths). In various embodiments the diameter of the fibers may be less than about 20 microns or greater than about 500 microns. Preferably, the diameter of the fibers will be from about 60 microns to about 80 microns.
xe2x80x9cAboutxe2x80x9d, in this one context is intended to mean a range of from 1-10 microns, which includes the intermediate lengths within the range. It will be readily understood that xe2x80x9cintermediate lengthsxe2x80x9d, in this context, means any length between the quoted ranges, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500 range.
The inventors also contemplate that the matrix may be woven, non-woven, braided, knitted, or a combination of two or more such preparations. For example, potential applications such as artificial arteries may well use a combination of woven, non-woven and knitted preparations or a combination of all four preparations. In certain embodiments of the invention, braided compositions may find particular utility for use with tendons and ligaments. Such braiding may, for example, provide superior strength.
In certain embodiments of the invention, the fibers containing one or more therapeutic agents are distributed within the scaffold matrix in a defined nonhomogeneous pattern. In one embodiment, the fibers may comprise two or more subsets of fibers that differ in biodegradable polymer content. The fibers or subsets of fibers may comprise a plurality of co-axial biodegradable polymer layers.
In another embodiment of the present invention, the fibers or a subset of fibers, contain one or more therapeutic agents such that the concentration of the therapeutic agent or agents varies along the longitudinal axis of the fibers or subset of fibers. The concentration of the active agent or agents may vary linearly, exponentially or in any desired fashion, as a function of distance along the longitudinal axis of a fiber. The variation may be monodirectional, that is, the content of one or more therapeutic agents decreases from the first end of the fibers or subset of the fibers to the second end of the fibers or subset of the fibers. The content may also vary in a bidirection fashion, that is, the content of the therapeutic agent or agents increases from the first ends of the fibers or subset of the fibers to a maximum and then decreases towards the second ends of the fibers or subset of the fibers.
In certain embodiments of the present invention, a subset of fibers comprising the scaffold may contain no therapeutic agent. For fibers that contain one or more therapeutic agents, the agent or agents may include a growth factor, an immunodulator, a compound that promotes angiogenesis, a compound that inhibits angiogenesis, an anti-inflammatory compound, an antibiotic, a cytokine, an anti-coagulation agent, a procoagulation agent, a chemotactic agent, an agents that promotes apoptosis, an agent that inhibits apoptosis, a mitogenic agent, a radioactive agent, a contrast agent for imaging studies, a viral vector, a polynucleotide, therapeutic genes, DNA, RNA, a polypeptide, a glycosaminoglycan, a carbohydrate, a glycoprotein. The therapeutic agents may also include those drugs that are to be administered for long-term maintenance to patients such as cardiovascular drugs, including blood pressure, pacing, anti-arrhythmia, beta-blocking drugs, and calcium channel based drugs. Therapeutic agents of the present invention also include anti-tremor and other drugs for epilepsy or other movement disorders. These agents may also include long term medications such as contraceptives and fertility drugs. They could comprise neurologic agents such as dopamine and related drugs as well as psychological or other behavioral drugs. The therapeutic agents may also include chemical scavengers such as chelators, and antioxidants. Wherein the therapeutic agent promotes angiogenesis, that agent may be vascular endothelial growth factor. The therapeutic agents may be synthetic or natural drugs, proteins, DNA, RNA, or cells (genetically altered or not). As used in the specification and claims, following long-standing patent law practice, the terms xe2x80x9caxe2x80x9d and xe2x80x9can,xe2x80x9d when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d or xe2x80x9cincludingxe2x80x9d means one or more.
In general, the present invention contemplates the use of any drug incorporated in the biodegradable polymer fibers of the invention. The word xe2x80x9cdrugxe2x80x9d as used herein is defined as a chemical capable of administration to an organism, which modifies or alters the organism""s physiology. More preferably the word xe2x80x9cdrugxe2x80x9d as used herein is defined as any substance intended for use in the treatment or prevention of disease. Drug includes synthetic and naturally occurring toxins and bioaffecting substances as well as recognized pharmaceuticals, such as those listed in xe2x80x9cThe Physicians Desk Reference,xe2x80x9d 471st edition, pages 101-321; xe2x80x9cGoodman and Gilman""s The Pharmacological Basis of Therapeuticsxe2x80x9d 8th Edition (1990), pages 84-1614 and 1655-1715; and xe2x80x9cThe United States Pharmacopeia, The National Formularyxe2x80x9d, USP XXII NF XVII (1990), the compounds of these references being herein incorporated by reference. The term xe2x80x9cdrugxe2x80x9d also includes compounds that have the indicated properties that are not yet discovered or available in the U.S. The term xe2x80x9cdrugxe2x80x9d includes pro-active, activated, and metabolized forms of drugs.
The biodegradable polymer may be a single polymer or a co-polymer or blend of polymers and may comprise poly(L-lactic acid), poly(DL-lactic acid), polycaprolactone, poly(glycolic acid), polyanhydride, chitosan, or sulfonated chitosan, or natural polymers or polypeptides, such as reconstituted collagen or spider silk.
One aspect of the present invention is a drug-delivery fiber composition comprising a biodegradable polymer fiber containing one or more therapeutic agents. In one embodiment, the content of the one or more therapeutic agents within the fiber varies along the longitudinal axis of the fiber such that the content of the therapeutic agent or agents decreases from the first end of the fiber to the second end of the fiber. In another embodiment, the fiber comprises a plurality of co-axial layers of biodegradable polymers. The drug delivery fiber composition may be implanted into many sites in the body including dermal tissues, cardiac tissue, soft tissues, nerves, bones, and the eye. Ocular implantation has particular use for treatment of cataracts, diabetically induced proliferative retinopathy and non-proliferative retinopathy, glaucoma, macular degeneration, and pigmentosa XXXX.
Another aspect of the present invention is a method of controlling the spatial and temporal concentration of one or more therapeutic agents within a fiber-scaffold implant, comprising implanting a fiber-scaffold into a host. The spatial concentrations may be provided across multiple fibers, or alternatively along a single fiber by imposing a concentration gradient along the length of a fiber. The fiber-scaffold typically comprises biodegradable polymer fibers containing one or more therapeutic agents, wherein the therapeutic agent or agents are distributed in the fiber-scaffold in a defined nonhomogeneous pattern. The host will typically be an animal, preferably a mammal and more preferably a human.
Yet another aspect of the present invention is a method of producing a fiber-scaffold for preparing an implant capable of controlling the spatial and temporal concentration of one or more therapeutic agents. This method generally comprises forming biodegradable polymer fibers into a three dimensional fiber-scaffold. The biodegradable polymer fibers contain one or more therapeutic agents. The therapeutic agent or agents are distributed in the fiber-scaffold in a defined nonhomogeneous pattern.
It is further envisioned that the scaffold of the invention may be used to direct and/or organize tissue structure, cell migration and matrix deposition and participate in or promote general wound healing.
In another embodiment of the invention, a method is provided for creating a drug releasing fiber from chitosan comprising use of hydrochloric acid as a solvent and Tris base as a coagulating bath. The hydrochloric acid concentration may be, for example, from about 0.25% to about 5%, or from about 1% to about 2%, including all concentrations within such ranges. In the method, the tris base concentration may be, for example, from about 2% to about 25%, from about 4% to about 17%, or from about 5% to about 15%, including all concentrations within such ranges. The method may, in one embodiment of the invention, comprise a heterogeneous mixture comprising chitosans with different degrees of deacetylation. The method may also comprise creating a drug releasing fiber comprising segments of chitosan with different degrees of deacetylation.
A drug releasing fiber in accordance with the invention may be created, for example, from chitosan and extracellular matrix. In creating a drug releasing fiber in accordance with the invention, the chitosan concentration may be, for example, from about 0.5 wt. % to about 10 wt. %, from about 1 wt. % to about 7 wt. %, from about 2 wt. % to about 5 wt. %, from about 3 wt. % to about 4 wt. %, or about 3.5 wt. %. In one embodiment of the invention, the Matrigel. The extracellular matrix concentration may be from about 1 vol. % to about 20 vol. %, from about 2 vol. % to about 15 vol. %, from about 3 vol. % to about 10 vol. %, or from about 4 vol. % to about 6 vol. %, including about 5 vol. %. In the method, the fiber may be coated with said extracellular matrix.
Chitosan used in accordance with the invention may be sulfated or unsulfated. In one embodiment of the invention, when sulfated chitosan is used the concentration may be from about 0.025 wt. % to about 2 wt. %, from about 0.05 wt. % to about 1 wt. %, from about 0.1 wt. % to about 0.5 wt. %, or from about 0.15 wt. % to about 0.3 wt. %, including about 0.2 wt. %. In the method, chitosan and sulfated chitosan may be extruded into a fiber.
In still another embodiment of the invention, a method is provided of creating a drug releasing fiber, the method comprising adding poly(L-lactic acid) microspheres to chitosan in acid and a coagulation bath. In the method, the acid may be, for example, acetic acid or hydrochloric acid. Where the acid is hydrochloric acid, the concentration may be, for example, from about 0.25% to about 5%, or from about 1% to about 2%, including 1.2 vol. % and all other concentrations within such ranges. The chitosan concentration may be, for example, from about 0.5 wt. % to about 10 wt. %, from about 1 wt. % to about 7 wt. %, from about 2 wt. % to about 5 wt. %, from about 3 wt. % to about 4 wt. %, or about 3.5 wt. %. The coagulation bath may comprise sodium hydroxide, for example, in a concentration of about 1 vol. % to about 20 vol. %, 2 vol. % to about 15 vol. %, 3 vol. % to about 10 vol. %, 4 vol. % to about 7 vol. %, or about 4 vol. % to about 6 vol. %, including about 5 vol. %. In one embodiment of the invention, the method comprises adding poly(L-lactic acid) microspheres to a solution of about 3.5 wt. % chitosan in from about 1 vol. % hydrochloric acid to about 2 vol. % hydrochloric acid and using a coagulation bath comprising from about 5 vol. % tris base to about 15 vol. % tris base. The method may further comprise adding a surfactant to the solution, including albumin, for example, from about 1 wt. % to about 5 wt. % of said albumin, including about 3 wt. %. In yet another embodiment of the invention, a composition of chitosan fibers is provided comprising microspheres of a second polymer, said microspheres comprising one or more biological molecules. The composition may comprise a surfactant that is a biological molecule.
In yet another embodiment of the invention, a composition is provided comprising a fiber containing chitosan and an extracellular matrix. The chitosan may be sulfated or non-sulfated.
In yet another embodiment of the invention, a composition is provided comprising a three-dimensional scaffold, said scaffold comprising fibers that are woven, non-woven, or knitted, wherein said fibers comprise any of the compositions described herein above. A composition in accordance with the invention may, in one embodiment, comprise fibers containing chitosan, extracellular matrix and a biological molecule. The chitosan may sulfated non-sulfated.
In yet another embodiment of the invention, a composition is provided comprising a heterogeneous scaffold of fibers a biological molecule as described above, wherein the biological molecule not the same for all fibers of the scaffold. In the composition, the degree of deacetylation may vary as a function of distance along the fiber. The composition may an extracellular matrix. The composition may also, in certain embodiments of the invention, comprise sulfated or non-sulfated chitosan.